Modulation of tor

ABSTRACT

Methods for screening for potential modulators of mammalian target of rapamycin (mTOR) are provided based on detecting cellular ATP levels. Also provided are compositions useful in treating diseases or conditions dependent on mTOR signaling, including cancer, rheumatoid arthritis, restinosis and transplant rejection.

The present invention relates to the field of tumour and cancer therapy.More particularly, the invention relates to methods of screening agentsfor anti-tumour and/or anti-tumourigenic activity, and therapies whichare intended to selectively kill or reduce the growth, division orviability of tumour or cancer cells compared to non-tumour or non-cancercells. The invention relates to the fields of molecular biology, cellbiology and pharmacology.

In the more affluent countries of the world cancer is the cause of deathof roughly one person in five. The American Cancer Society in 1993reported that the five most common cancers are those of the lung,stomach, breast, colon/rectum and the uterine cervix. Tumour cells havelost the normal control of the cell cycle and so divide out of controlcompared to normal cells. The sub-cellular machinery which controlscellular processes is made up of a complex biochemical network ofinteracting proteins that induce and co-ordinate the essential processesof cell growth, duplication and division.

Environmental cues are deciphered by cellular regulatory elements toadjust the metabolic state within the cell to reflect externalconditions and maintain cellular homeostasis. The mammalian target ofrapamycin (mTOR or TOR) is a member of the phosphatidylinositide kinaserelated family of protein kinases and resides at the interface betweennutrient sensing and the regulation of major metabolic responses (Denniset al., Curr Opin Genet & Dev 9, 49 (1999); Gingras et al., Genes & Dev.15, 807 (2001); Schmelzle and Hall, Cell 103, 193 (2000)). Morespecifically, depending on mitogen and amino acid availability, mTORpositively regulates translation and ribosome biogenesis whilenegatively controlling autophagy (Dennis et al., 1999), leading to thesuggestion that mTOR acts to set protein synthetic rates as a functionof the availability of translational precursors (Hara et al., J. Biol.Chem. 273,14484 (1998); liboshi et al., J.Biol.Chem. 274,1092 (1999)).In response to mitogens and amino acids, mTOR phosphorylates andcontrols the activities of two key translational regulators, S6 Kinase 1(S6K1) and initiation factor 4E binding protein (4E-BP1) (Gingras etal., 2001). However, the ability to detect changes in mTOR activity invitro, following either mitogen or amino acid treatment, has beendifficult to demonstrate (Gingras, et al., 2001; Schmelzle and Hall,2000).

The importance of understanding the molecular mechanisms that controlmTOR function is underscored by recent Phase 1 clinical trials showingthat rapamycin is efficacious in the treatment of solid tumors inpatients with metastatic renal cell carcinoma, and non-small cell lung,prostate, and breast cancer (Hidalgo and Rowinsky, 2000, Oncogene 19,6680). Nevertheless, there remains a need to find alternative ways ofselectively killing a wide range of cancer cells whilst leaving normalcells of the body unaffected.

RELEVANT LITERATURE

-   P. B. Dennis, S. Fumagalli, G. Thomas, Curr Opin Genet & Dev 9, 49    (1999).-   A. C. Gingras, B. Raught, N. Sonenberg, Genes & Dev. 15, 807 (2001).-   T. Schmelzle, M. N. Hall, Cell 103, 193 (2000).-   K. Hara et al., J. Biol. Chem. 273, 14484 (1998).-   M. Hidalgo, E. K. Rowinsky, Oncogene 19, 6680 (2000).-   E. V. Schmidt, Oncogene 18, 2988 (1999).-   J. Roberts, Science 278, 2073 (1997).-   C. J. Lynch, H. L. Fox, T. C. Vary, L. S. Jefferson, S. R.    Kimball, J. Cell. Biochem. 77, 234 (2000).-   T. Gaal, M. S. Bartlett, W. Ross, C. L. Turnbough, Jr., R. L.    Gourse, Science 278, 2092 (1997).

SUMMARY OF THE INVENTION

The present invention provides a method for screening for a potentialmodulator of TOR comprising: incubating a test agent with a cell;detecting a decrease in ATP levels in the cell relative to when the testagent is absent; and correlating a decrease in ATP levels in the cellwith the presence of a potential modulator of TOR. The decrease in ATPlevels is at least 1% preferably, at least 5-10%, but no more than 75%,preferably 50%. ATP levels in a cell can be conventiently detected usingluciferase-based assays.

The potential modulator is preferably effective against a disease orcondition dependent on mTOR signaling, including without limitationcancer, rheumatoid arthritis, restinosis and transplantation rejection,and therefore can be used for the treatment, including the prophylactictreatment of these diseases and conditions.

The invention therefore also provides compositions for the prevention orprophylactic treatment of tumourigenesis or the treatment orprophylactic treatment of tumours, rheumatoid arthritis (or otherinflammatory diseases), restinosis, transplant rejection or thetreatment or prophylactic treatment of any disease involving mTOR,comprising a compound that reduces ATP levels in a cell by at least atleast 1% preferably, at least 5-10%. The compound preferably does notreduce ATP levels in a cell by more than 75%. The composition may beprovided together with a pharmaceutically acceptable excipient, diluentor carrier, for use as a pharmaceutical.

Also encompassed by the invention is the use of such compositions forthe manufacture of a pharmaceutical or for the treatment of a disease orcondition dependent on mTOR, or for the treatment of cancer, rheumatoidarthritis, restinosis or transplant rejection. The cancer will typicallybe characterized by having high intracellular ATP concentrations. Thecancer may be a solid tumor. The cancer may be epithelial ormesenchymal, such as a glioblastoma or breast carcinoma.

Also provided are methods of treating a disease or condition dependenton mTOR, comprising administering an effective amount of a compound thatreduces ATP levels in a cell by at least at least 1% preferably, atleast 5-10%, but not more than 75%. The disease or condition can beselected from the group consisting of: cancer, rheumatoid arthritis,restinosis and transplant rejection.

In a further aspect of the invention, a method of diagnosing orprognosing a disease or condition dependent on mTOR is provided. Themethod comprises obtaining a sample from an individual; analysing thesample for the presence of ATP or an ATP marker (such as a glycolyticenzyme); and correlating the presence of an elevated level of ATP or theATP marker relative to a sample from an unafflicted individual with anunfavourable prognosis or diagnosis.

DETAILED DESCRIPTION

The bacterial macrolide, rapamycin is an efficacious anti-cancer agentagainst solid tumors. In a hypoxic environment the increase in mass ofsuch tumors is dependent on the recruitment of mitogens and nutrients.When nutrient concentrations change, particularly those of essentialamino acids, the mammalian Target Of Rapamycin (mTOR), functions inregulatory pathways that control ribosome biogenesis and cell growth.The present invention is based on the observation that the mTOR pathwayis influenced by the intracellular concentration of ATP, independent ofthe abundance of amino acids, and that mTOR itself is an ATP sensor. Itis proposed that as ATP is utilized in eukaryotic cells, mTOR functionsas a homeostatic sensor, adjusting the rate of ribosome biogenesis toreflect intracellular ATP concentrations.

In tumors metabolic flux is redirected to glycolysis, leading to themore rapid production of ATP. The present inventors believe that suchtumours having an increased production of ATP can be more susceptible tothe effects of mTOR inhibitors and more sensitive to reduction inintracellular ATP concentrations, compared to normal cells.

Accordingly, the present invention provides a method for screening for apotential modulator of mTOR signalling comprising: incubating a testagent with a cell, detecting a decrease in ATP levels in said cellrelative to when said test agent Is absent; and correlating a decreasein ATP levels in said cell with the presence of a potential modulator ofmTOR signalling. The cell is preferably a mammalian cell, morepreferably a human cell, and typically will be available as a cell linefor ease of propagation.

The test agent can be present in a library of compounds, which can betested in pools to reduce the time needed to identify a potentialmodulator. Once a potential modulator is identified as being present Ina pool of test agents, each individual test agent can be re-tested toidentify the potential modulator. Alternatively, known compounds can betested without pooling and chemically modified to obtain or improve thedesired property. For example, known ATP depleting agents include2-deoxyglucose, cyanine, oligomycin, valinomycin and azide, as well assalts and derivatives thereof. Such ATP depleting agents can bechemically modified or provided in formulations to give the desiredeffect, i.e., a decrease in intracellular ATP levels sufficient toaffect mTOR signaling but without having a detrimental effect on normalcells.

Typically, useful modulators will result in a decrease of at least 1% inintracellular ATP levels, preferably at least 5%, more preferably atleast 15% or more. To avoid detrimental effects on normal cells, thedecrease is preferably no more than 75%, most preferably no more than50%.

ATP levels can be determined by any method known in the art or anymethod yet to be discovered. Examples of methods that can be used todetermine ATP levels are described in the Examples below, which use aluciferase assay to detect ATP, as well as in U.S. Pat. No. 5,618,682and WO 00/18953, which are hereby incorporated by reference in theirentirety.

The screening methods of the Invention may further comprise controlcells grown in the absence of test agent and ATP levels are measured inboth control and test cultures. The test measurements can thereby benormalized with respect to the control. Other internal controls can alsobe employed to test for reproducibility of the assay or any otherdesired characteristic, as is well known in the art.

Throughout the assays of the invention, incubation and/or washing stepsmay be required after each application of reagent or incubation ofcombinations of reagents. Incubation steps may vary from about 5 minutesto several hours, perhaps from about 30 minutes to about 6 hours.However, the incubation time usually depends upon the assay format,analyte, volume of solution, concentrations, and so forth. The assayscan be carried out at ambient temperature, although they may also beconducted at temperatures in the range of 4° C. to 40° C., for example.Assays with cell extracts are typically carried out at 4° C.

mTOR is known to play a pivotal role in a number of diseases andconditions. Thus, the potential modulator can be further tested for itseffectiveness against any disease or condition dependent on mTORsignaling, such as by determining the effect of the potential modulatoron mTOR kinase activity. The disease or condition includes withoutlimitation cancer, rheumatoid arthritis (or other inflammatorydiseases), restinosis (endothelial cell inhibition) and transplantationrejection. Thus, also included within the scope of the present inventionare potential modulators of mTOR signaling for the treatment orprophylactic treatment of cancer, rheumatoid arthritis, restinosis andprevention of transplant rejection or for the treatment or prophylactictreatment of any disease or condition involving mTOR signalling,identified by any of the screening methods of the invention. Thesesubstances may be proteins, polypeptides, natural compounds (e.g.,polyketides) or small organic molecules (drugs). The invention thereforeincludes pharmaceutical compositions for preventing or treating anydisease or condition involving mTOR signalling comprising one or more ofthe substances identified by a screening method of the invention.

Thus, in a further aspect of the invention, compositions are provided,in particular pharmaceutical compositions for humans or veterinarycompositions for animals, for the prevention or prophylactic treatmentof tumourigenesis or the treatment or prophylactic treatment of tumours(including mesenchymal or epithelial tumours, such as, withoutlimitation, glioblastoma, or leukemia (in particular abnormal T cellproliferation), lung, stomach, breast, colon/rectum, uterine or cervicalcancer), rheumatoid arthritis, restinosis, prevention of transplantrejection or the treatment or prophylactic treatment of any diseaseinvolving mTOR signalling, comprising a compound that reduces ATP levelsin a cell by at least at least 1% preferably, at least 5-10%, or atleast 25% but not more than 75%, preferably not more than 50%. Thecompositions preferably comprise a compound that affects mTOR activity.Thus, the compositions will typically be effective against cancers(e.g., solid tumours or cancers with high intracellular ATPconcentrations) that are rapamycin sensitive. The compositions may alsoinclude other active or non-active agents. Non-active agents may includea pharmaceutically acceptable excipient; diluent or carrier, such assaline, buffered saline, dextrose or water.

The present invention further provides the use of a potential modulatorof mTOR signalling as hereinbefore, for the manufacture of a medicamentfor the prevention or prophylactic treatment of a disease or conditiondependent on mTOR signaling, such as cancer, rheumatoid arthritis,restinosis or prevention of transplantation rejection(immunosuppression).

The compositions and medicaments of the invention may therefore be usedprophylactically in order to prevent tumours or other diseases fromdeveloping, or they may be used in a curative or partly curative way totreat or contain a pre-existing condition. The tumours or tumour cellsare preferably those which are sensitive to rapamycin and/or those whichexhibit a high intracellular ATP concentration. In particularlypreferred embodiments the tumours are solid tumours, e.g. mesenchymaltumours such as glioblastoma or epithelial cancers such as breastcarcinoma.

The invention also provides a method of treating a disease or conditiondependent on mTOR, comprising administering an effective amount of acompound that reduces ATP levels in a cell by at least at least 1%preferably, at least 5-10%, at least 25%, but not more than 75%,preferably not more than 50%. The term “treating” is meant to encompassprophylactic treatment as well as the treatment of an existing diseaseor condition. The disease or condition includes without limitation,cancer, rheumatoid arthritis, restinosis and prevention of transplantrejection.

The determination of an effective dose Is well within the capability ofthose skilled in the art. For any compound, the therapeuticallyeffective dose can be estimated initially either in cell culture assaysor in an appropriate animal model. The animal model is also used toachieve a desirable concentration range and route of administration.Such information can then be used to determine useful doses and routesfor administration in humans.

A therapeutically effective dose refers to that amount of active agentwhich ameliorates the symptoms or condition. Therapeutic efficacy andtoxicity of such compounds can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals (e.g., ED50, thedose therapeutically effective in 50% of the population; and LD50, thedose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can beexpressed as the ratio, LD50/ED50. Pharmaceutical compositions whichexhibit large therapeutic indices are preferred. The data obtained fromcell culture assays and animal studies is used in formulating a range ofdosage for human use. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, sensitivity of the patient, and the routeof administration.

The exact dosage may be chosen by the individual physician in view ofthe patient to be treated. Dosage and administration can be adjusted toprovide sufficient levels of the active moiety or to maintain thedesired effect. Additional factors which may be taken into accountinclude the severity of the disease state (e.g. tumour size andlocation); age, weight and gender of the patient; diet; time andfrequency of administration; drug combination(s); reactionsensitivities; and tolerance/response to therapy. Long actingpharmaceutical compositions can be administered on a daily basis, every3 to 4 days, every week, or once every two weeks, depending on half-lifeand clearance rate of the particular formulation.

Administration of pharmaceutical compositions of the invention may beaccomplished orally or parenterally. Methods of parenteral deliveryinclude topical, intra-arterial (e.g. directly to the tumour),intramuscular, subcutaneous, intramedullary, intrathecal,intraventricular, Intravenous, intraperitoneal, or intranasaladministration. In addition to the active ingredients, thesepharmaceutical compositions can contain suitable pharmaceuticallyacceptable carriers comprising excipients and other compounds thatfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. Further details on techniques forformulation and administration can be found in the latest edition ofRemington's Pharmaceutical Sciences (Maack Publishing Co, Easton Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, etc, suitablefor ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable additional compounds, if desired, to obtaintablets or dragee cores. Suitable excipients are carbohydrate or proteinfillers include, but are not limited to sugars, including lactose,sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato,or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose., or sodium carboxymethylcellulose; andgums including arabic and tragacanth; as well as proteins such asgelatin and collagen. If desired, disintegrating or solubilizing agentsmay be added, such as the cross-linked polyvinyl pyrrolidone, agar,alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores can be provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterise the quantity ofactive compound (i.e. dosage).

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds can bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions can contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active compoundscan be prepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes. Optionally, the suspension can also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention can bemanufactured in substantial accordance with standard manufacturingprocedures known in the art (e.g. by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilising processes).

The present invention also provides a method of diagnosing or prognosinga disease or condition dependent on mTOR, comprising: obtaining a samplefrom an individual; analysing the sample for the presence of a markerindicative of ATP levels in the sample; and correlating the presence (oran increase in the level of the marker relative to a value obtained froman unafflicted individual) with an unfavourable prognosis or diagnosis.Such a marker is easily selected by one of ordinary skill in the art andincludes glycolytic enzymes or their activity, for example. Astatistically significant increase in the presence of such a marker, orits activity, relative to normal tissue would therefore indicate anunfavourable prognosis or diagnosis. The intracellular ATP concentrationcould itself act as such a marker and as an indicator of an individualhaving a higher risk of a disease or condition dependent on mTORsignaling.

In a preferred embodiment, the invention provides kits suitable for usein the diagnostic or prognostic methods of the invention. Such kitscomprise reagents useful for carrying out these methods, for example, anantibody specific for the marker (e.g., a glycolytic enzyme), reagentsuseful in detecting glycolytic enzymatic activity, or detecting ATP,such as luciferase.

Preferred embodiments of the invention will now be described by way ofexample, which should not be considered limiting in any way.

EXAMPLES Example 1 mTOR is Sensitive to Metabolic Inhibitors

Because mTOR is sensitive to amino acids and regulates ribosomebiogenesis (Dennis et al., 1999; Gingras et al., 2001; Schmelzle andHall, 2000), we tested whether its activity may be sensitive tometabolic inhibitors. We used, insulin-induced S6K1 activation and4E-BP1 phosphorylation as reporters for mTOR function in the presence ofglycolytic or mitochohdrial inhibitors to reduce ATP production.

Briefly, human embryonic kidney cells (HEK293) were seeded andmaintained as previously described (Pullen et al., Science 279, 707(1998)). Confluent cells were serum starved for 20 h and then extracted(controls) or treated with 200 nM insulin in the precence or absence of100 mM 2-deoxyglucose or 20 mM rotenone for 30 min. Cell extraction,kinase assays and Western blot analysis (used to measure S6K1 levels andT389 phosphorylation) were performed as described (Pullen et al.,1998).Phosphospecific antibodies are commercially available (e.g., New EnglandBiolabs). Although an inhibitory effect was seen with rotenone, theglycolytic inhibitor 2-deoxyglucose (2-DG) was shown to be moreeffective in inhibiting S6K1 T389 phosphorylation and S6K1 activation(determined by detecting S6 phosphorylation) than the mitochondrialinhibitor, rotenone, reducing phosphorylation/activation to backgroundlevels.

The results were confirmed using a different target of mTOR, namely4EBP-1. Transfections were carried out essentially as described byManteuffel et al., 1997, Mol Cell. Biol. 17, 5426 with the 4EBP-1construct described therein. The expression and phosphorylation oftransiently-transfected HA-4E-BP1 was measured using a polyclonalanti-HA antibody and phosphospecific antibody against S65, respectively,essentially as described above (Pullen et al., 1998). The anti-HAantibody was obtained from a commercial source (Santa CruzBiotechnology, Inc). The mTOR-mediated phosphorylation site in 4E-BP1 isS65. Western blot analysis showed an inhibitory effect of rotenone onS65 phosphorylation in insulin stimulated cells, whereas the glycolyticinhibitor 2-deoxyglucose (2-DG) was again more effective, asdemonstrated by its ability to inhibit 4EBP-1 S65 phosphorylation tobackground levels. Similar results were obtained when phosphorylation ofthe S65 site was measured after treatment with iodoacetic acid ordinitrophenol, known glycolytic or mitochondrial inhibitors,respectively (i.e., iodoacetic acid was found to be more efficient thandinitrophenol).

In summary, the metabolic inhibitors iodoacetic acid, dinitrophenol,2-deoxyglucose and rotenone are all able to inhibit phosphorylation ofmTOR targets. However, only the glycolytic inhibitors (2-deoxyglucoseand lodoacetic acid) are able to reduce phosphorylation of mTOR targetsto background levels.

Example 2 mTOR is Sensitive to Alterations in Intracellular ATP

Because mTOR is sensitive to glycolitic inhibitors, we wanted todetermine whether mTOR is sensitive to alterations in intracellular ATPconcentrations. ATP levels were measured from mock-transfected HEK293cells (using empty vector) treated as in Example 1, using aluciferase-based assay. Retonone and 2-deoxyglucose treatments werecarried out on insulin-stimulated cells.

To generate extracts for ATP assays, cells were washed twice with 10 mlice cold PBS, drained thoroughly, scraped into 1 ml of buffer (100 mMTris-HCl and 4 mM EDTA pH 7.75) and transferred into an Eppendorf tubebefore flash freezing in liquid nitrogen. The frozen cells were boiledfor 3 min and then placed on ice for 5 min followed by centrifugation at13,000 rpm for 5 min at 4° C. ATP levels in the extract were measured ina microtiter plate by a Luciferase-based assay (Roche, ATPBioluminescence Assay Kit CLS II) using a Microlumat LB96P microtiterplate reader (EG&G Berthold). In an illustrative set of experiments andexpressing results as a percentage of the insulin-stimulated control(100%), ATP levels increased slightly in insulin treated cells (94% inunstimulated cells), decreased in 2-deoxyglucose treated cells (40-49%)and rotenone-treated cells (75-83%). The ability of each agent to lowerATP concentrations therefore paralleled its effectiveness in inhibitingS6K1 activation suggesting that mTOR is sensitive to alterations inintracellular ATP.

Example 3 Selectivity of Metabolic Inhibitors

The modest effect of rotenone in reducing ATP concentrations, ascompared to its effects on S6K1 and 4E-BP1 phosphorylation, suggestedthe metabolic inhibitors were not generally toxic. To verify this, theeffect of 2-deoxyglucose was tested on Protein Kinase B (PKB) andMitogen-Activated Protein Kinase (MAPK).

Transiently-transfected, HA-tagged PKB activation was measured in vitrousing histone 2B (H2B) as substrate (Franke et al., 1995, Cell 81, 727)after immunoprecipitation from serum-starved cells extracted directly orafter insulin stimulation with or without the addition of 100 mM2-deoxyglucose, 20 nM rapamycin or 100 nM wortmannin. Rapamycin andwortmannin were added to serum-starved cells 30 min prior toinsulin-stimulation. HA-PKB expression and S473 phosphorylation weremeasured essentially as described above. HA-MAPK kinase activity towardusing myelin basic protein (MBP) was measured from serum-starved cellsextracted directly or after 100 nM TPA stimulation in the presence orabsence of 20 nM rapamycin or 100 mM 2-deoxyglucose for 30 min (N.Pullen et al., 1998).

Like rapamycin, 2-deoxyglucose did not inhibit insulin-inducedactivation of PKB, as judged by S473 phosphorylation and in vitrophosphorylation of H2B. However, both S473 and H2B phosphorylation wereshown to be sensitive to wortmannin, a phosphatidylinositide-3OH kinase(PI3K) inhibitor. In addition, 2-deoxyglucose had no discernable effecton TPA-induced MAPK activation, as judged by MBP phosphorylation. Hence,the effect of 2-deoxyglucose in reducing ATP concentrations is selectivefor signaling to S6K1 and 4E-BP1.

Example 4 Specific Inhibition of mTOR-Signalling by 2-deoxyglucose

To assess whether the inhibitory effects of 2-deoxyglucose on S6K1activation were mediated by mTOR, we used a rapamycin-resistant alleleof S6K1. Rapamycin resistance was conferred by fusingglutathione-S-transferase to the NH₂-terminus of S6K1 and truncating theCOOH-terminus, creating a construct termed GST-ΔC-S6K1 (Pullen et al.,1998). For the construction of the GST-ΔC-S6K1 (also known asGST-ΔC₁₀₄-S6K1) and D3E, K100Q-S6K1-GST plasmids, similar cloningstrategies were used. S6K1-GST or GST-ΔC-S6K1 fromtransiently-transfected, serum-starved HEK293 cells were extracteddirectly or after insulin stimulation with or without the addition of 20nM rapamycin or 100 nM wortmannin. Kinase activities, expression levelsand T389 phosphorylation were carried out essentially as describedabove. Expression and activity of S6K1-GST or GST-ΔC-S6K1 were measuredafter insulin stimulation alone or with increasing amounts of2-deoxyglucose (20, 40 and 100 mM) essentially as described above.Mock-transfected HEK293 cells were treated using the same conditions andextracted for ATP analysis

Both S6K1, having a COOH-terminal GST tag (S6K1-GST), and GST-ΔC-S6K1were phosphorylated and activated by insulin in a wortmannin-sensitivemanner, however only GST-ΔC-S6K1 was resistant to inhibition byrapamycin. 2-deoxyglucose reduced insulin-induced activation of S6K1-GST(rapamycin sensitive) in a dose-dependent manner, paralleling its effecton intracellular ATP concentrations. Compared to ATP levels in anInsulin stimulated control (100%), ATP levels decreased to 57-59%,43-48% and 32-36% after treatment of S6K1-GST transfected cells with 20,40 and 100 nM 2-deoxyglucose, respectively. In contrast, insulin-inducedactivation of GST-ΔC-S6K1 (rapamycin-resistant) was unaffected by2-deoxyglucose treatment. Thus, 2-DG appears to selectively inhibitsignaling to mTOR effectors, supporting a model whereby mTOR iscontrolled by intracellular ATP concentrations.

Example 5 lntracellular ATP Levels have a Direct Effect on mTOR Activity

Because mTOR signaling Is dependent on concentrations of aminoacylatedtRNAs (liboshi et al., 1999, J.Biol.Chem. 274,1092), the effects of ATPon S6K1 and 4E-BP1 may be indirect, occurring through inhibition of tRNAaminoacylation. To examine this possibility, we analyzed total cellulartRNA on acid-urea polyacrylamide gels, which resolve aminoacylated fromnon-acylated tRNA.

Briefly, total RNA was isolated from serum-starved HEK293 cells directlyor after insulin stimulation with or without 2-deoxyglucose or aminoacid starvation. As a control marker, tRNA was deacetylated by mildalkaline hydrolysis in 0.1 M Tris-HCl (pH 8.0) at 75° C. for 5 min.After resolution on an acid/urea gel (Enriquez et al., in MethodsEnzymol., Acad. Press, San Diego, vol. 264, pp. 183.), tRNA wasvisualized with ethidium bromide staining. Expression levels andphosphorylation state of endogenous S6K1 and 4E-BP1 were also measuredfrom serum-starved cells, stimulated with insulin in the presence orabsence of amino acids in the media essentially as described in Example1.

Neither insulin stimulation nor 2-deoxyglucose treatment had an effecton total amounts of aminoacylated tRNA. Unexpectedly, amino aciddeprivation also had no effect, even though such treatment wassufficient to completely block phosphorylation of S6K1 and 4E-BP1.

To further test this finding, selected tRNAs were examined by Northernblot analysis of the tRNA resolved by acid/urea gel electrophoresis asdescribed above. The individual tRNA species were probed afterelectrotransfer to nylon membrane, with radiolabelled oligonucleotidesspecific for tRNALeu and tRNAHis, namely: tRNALeu 5′-GCG CCT TAG ACC GCTCGG CCA CG-3′ (SEQ ID NO:1); tRNAHis 5′-GGT GCC GTG ACT CGG ATT CGA ACCG-3′ (SEQ ID NO:2); and tRNAThr 5′-GCG AGA AAT GM CTC GCG-3′ (SEQ IDNO:3). As with total tRNA, none of the treatments had an effect on theaminoacylation status of leucyl, histidyl or threonyl tRNA, indicatingthat amino acid pools, rather than amounts of aminoacylated tRNA, areimportant for mTOR signaling.

Levels of individual amino acids were measured in extracts prepared frominsulin-stimulated HEK293 cells in the presence or absence of aminoacids or with 100 mM 2-deoxyglucose treatment and expressed as apercentage of the insulin-stimulated control in the presence of aminoacids. Briefly, confluent cultures of HEK293 cells were treated asdescribed above, washed with PBS and drained thoroughly. The cells werethen scraped into 500 ml water and sonicated with four 10 sec pulses.Cell debris was removed by centrifugation and sulfosalicylic acid wasadded to the supernatant to a final concentration of 2%. The sampleswere then placed on ice for 30 min followed by centrifugation to removeprecipitated proteins. The extracts were then analyzed for amino acidcontent with a Biochrom 20 plus amino acid analyzer (Amersham-PharmaciaBiotech).

Amino acid deprivation was demonstrated to result in a decrease in theamounts of essential amino acids, particularly the branched-chain aminoacids. For example, Leu, Ile and Val levels decreased to 10-20% of thecontrol. In contrast, 2-deoxyglucose had little effect on amino acidlevels (Ala, Glu, Leu, Ile and Val, all 100-120%). Furthermore, aminoacid deprivation had no effect on concentrations of ATP. Thus,regulation of mTOR by ATP is independent of amino acids pools.

Example 6 mTOR Activity Requires High ATP Concentrations.

Reducing ATP concentrations could lead to a stable change in mTORactivity, through a post-translational modification, or ATP coulddirectly affect mTOR activity. To test the first possibility, wetransiently expressed HA epitope-tagged mTOR in HEK293 and measured itsactivity after treatment of cells with 2-deoxyglucose.

Briefly, transiently transfected, hemaglutinin-tagged (HA) wild-typemTOR (HA-mTORwt) from HEK293 cells were serum starved for 16 hoursbefore insulin stimulation. The cells were then treated with (orcontrols without) 100 mM 2-deoxyglucose or subjected to amino acidstarvation as described above. Cell extracts were prepared forimmunoprecipitations with a monoclonal anti-HA antibody. Theimmunocomplex was washed once with 1 M NaCl in assay buffer (30 mM MOPS(pH7.5), 5 mM NaF, 20 mM β-glycerol phosphate, 1 mM dithioerythritol,0.1% Triton X-100 and 10% glycerol) and twice with assay buffer alone.HA-mTORwt was assayed for T389 kinase activity, using a kinase inactivemutant of S6K1 (D3E, Pearson et al., 1995, EMBO J. 14, 5279; K100Q-GST,von Manteuffel et al., 1997, Mol. Cell. Biol. 17, 5426) as substrate.One microgram of a kinase inactive, purified, soluble S6K1 substrate(S6K1-D3E,K100 Q-GST) was added along with assay buffer containing 10 mMMgCl₂ and 1 mM ATP and incubated for 30 min at 30° C. Quantitation forKm measurements was carried out using scanning densitometry andImageQuant software (Molecular Dynamics).

To test whether an activated allele of P13K could affect mTOR activityin vitro, HA-mTORwt was transiently expressed alone or with aconstitutively membrane-targeted phosphatidylinositide-3-OH kinase(CD2-P13K, Reif et al., 1997, J. Biol. Chem. 272, 14426) using standardtechniques and then extracted directly or stimulated with insulin beforeextraction essentially as described above.

Although 2-deoxyglucose treatment blocked insulin-induced activation ofS6K1 (see Example 1), it had no effect on the kinase activity of mTOR invitro towards T389 of S6K1. Indeed, amino acid deprivation, insulinstimulation, or transient expression of an activated-allele of P13K hadno effect on mTOR kinase activity in vitro. Thus, it seems unlikely thata post-translational modification directly affects mTOR activity.

As a control, Myc-tagged, wild-type S6K1 was expressed alone or withCD2-P13K in starved or insulin-stimulated HEK293 cells. S6K1 kinaseactivity was assayed as in Example 1 after immunoprecipitation with ananti-myc antibody and Cd2-P13K, as well as insulin, CD″-P13K was shownto be able to induce T389 phosphorylation and S6K1 activation in intactcells.

Next, we measured mTOR activity in vitro at ATP concentrations thatapproached physiological levels of 1-5 mM In mammalian cells (Gribble etal., 2000, J.Biol.Chem. 275, 30046). The expression and activities ofHA-mTORwt and inactive mTOR kinase were assayed using S6K1 (T389phosphorylation) or 4E-BP1 (S65 phosphorylation) as substratesessentially as described above. ATP concentrations used in the assaywere 3.0 mM for inactive kinase and 0.2, 0.4, 0.8, 1.6, 2.3 and 3.0 mMfor wild-type mTOR.

Specific activity of mTOR for S6K1 T389 phosphorylation increased up to˜1 mM ATP, saturating at around 2-3 mM ATP, whereas the catalyticallyinactive mTOR mutant did not phosphorylate T389. On the basis of thesevalues we calculated an apparent Km for ATP of at least 1 mM. Mostprotein kinases analyzed to date show an apparent Km for ATP of 10-20 mM(Edelman et al., in Annu.Rev.Biochem., Annual Reviews Inc., Palo Alto,1987, vol. 56, pp. 567), one fiftieth to one hundredth of that observedfor mTOR. Because mTOR also phosphorylates several sites in 4E-BP1,including S65, we also assayed the ATP requirement of mTOR for S65 of4E-BP1. Using the same assay conditions described for T389phosphorylation in S6K1, we obtained almost identical results for S65phosphorylation. These findings sustain the role of mTOR as an ATPeffector, and suggest it is a direct sensor of ATP in the cell.Therefore, intracellular concentrations of ATP directly regulates mTOR,whereas amino acids employ a separate mechanism

Without wishing to be bound by theory, the inventors propose that as ATPis utilized in eukaryotic cells, mTOR functions as a homeostatic sensor,adjusting the rate of ribosome biogenesis to reflect intracellular ATPconcentrations. Interestingly, increased ribosome biogenesis is apredictive indicator of tumor progression (Derenzini et al., 2000, J.Pathol. 191, 181) and in tumors metabolic flux is redirected toglycolysis, leading to the more rapid production of ATP (Pfeiffer etal., 2001, Science 292, 504 (2001); Dang et al., 1999, Trends Biochem.Sci. 24, 68). If such tumors gain an mTOR-specific growth advantage, dueto increased production of ATP, they may be more susceptible to theeffects of mTOR inhibitors. Because of the low Km of mTOR for ATP (1mM), tumours may also be more sensitive to reduction in cellular ATPlevels. In contrast, most cellular kinases have a Km for ATP in therange of 10-20 μM and would remain unaffected by marginally reducedintracellular ATP concentrations.

The disclosures of each publication referred to herein are herebyincorporated by reference in their entireties, as if each reference werereferred to individually.

1. A method for screening for a potential modulator of TOR comprising:(a) incubating a test agent with a cell; (b) detecting a decrease in ATPlevels in said cell relative to when said test agent is absent; and (c)correlating a decrease in ATP levels in said cell with the presence of apotential modulator of TOR.
 2. The method of claim 1, wherein saiddecrease is at least 1% preferably, at least 105-10%.
 3. The method ofclaim 1, wherein said decrease is no more than 50%.
 4. The method ofclaim 1, wherein said ATP levels in said cell are detected using aluciferase assay.
 5. The method of claim 1, wherein said potentialmodulator is effective against a disease or condition dependent on mTORsignaling.
 6. The method of claim 1, wherein said potential modulator iseffective against any one of the diseases or conditions selected fromthe group consisting of: cancer, rheumatoid arthritis, restinosis andtransplantation rejection.
 7. A composition for the prevention orprophylactic treatment of tumourigenesis or the treatment orprophylactic treatment of tumours, rheumatoid arthritis, restinosis,transplant rejection or the treatment or prophylactic treatment of anydisease involving mTOR, comprising a compound that reduces ATP levels ina cell by at least at least 1% preferably, at least 5-10%, but not morethan 75%.
 8. The composition of claim 7, further comprising apharmaceutically acceptable excipient, diluent or carrier.
 9. Thecomposition of claim 7, wherein said compound affects mTOR activity. 10.The composition of claim 7, for use as a pharmaceutical.
 11. The use ofa composition of claim 7 for the treatment of cancer, rheumatoidarthritis, restinosis or transplant rejection.
 12. The use as claimed inclaim 11, wherein the cancer is rapamycin sensitive.
 13. The use asclaimed in claim 12, wherein the cancer is characterized by a highintracellular ATP concentration.
 14. The use as claimed in claim 12,wherein the cancer is mesenchymal or epithelial.
 15. The use as claimedin claim 12 wherein the cancer is a solid tumour.
 16. The use as claimedin claim 12, wherein the cancer is a glioblastoma or breast carcinoma.17. A method of treating a disease or condition dependent on mTOR,comprising administering an effective amount of a compound that reducesATP levels in a cell by at least at least 1% preferably, at least 5-10%,but not more than 75%.
 18. The method of claim 17, wherein said diseaseor condition is selected from the group consisting of: cancer,rheumatoid arthritis, restinosis and transplant rejection.
 19. A methodof diagnosing or prognosing a disease or condition dependent on mTC3R,said method comprising: a) obtaining a sample from an individual; b)analysing said sample for the presence ATP or an ATP marker; and c)correlating the presence of an elevated level of said ATP or ATP markerrelative to a sample from an unafflicted individual with an unfavourableprognosis or diagnosis.
 20. The method of claim 19, wherein said markeris a glycolytic enzyme.